Camptothecin-Binding Moiety Conjugates

ABSTRACT

The invention relates to therapeutic conjugates with improved ability to target various diseased cells containing a targeting moiety (such as an antibody or antibody fragment), a linker and a camptothecin as a therapeutic moiety, and further relates to processes for making and using the said conjugates.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/026,811, filed Feb. 6, 2008, which is a continuation-in-part of U.S.patent application Ser. No. 11/388,032, filed Mar. 23, 2006, whichclaimed the benefit under 35 USC 119(e) of provisional U.S. patentapplication Ser. Nos. 60/668,603, filed Apr. 6, 2005; 60/728,292, filedOct. 19, 2005 and 60/751,196, filed Dec. 16, 2005; and which was acontinuation-in-part of U.S. patent application Ser. No. 10/734,589,filed Dec. 15, 2003, which claimed the benefit under 35 USC 119(e) ofprovisional U.S. patent application Ser. No. 60/433,017, filed Dec. 13,2002. The text of each of the priority applications is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to therapeutic conjugates with improvedability to target various cancer cells, infectious disease organismsand/or for treating autoimmune diseases, which conjugates contain atargeting (binding) moiety and a therapeutic moiety belonging to thecamptothecin group of drugs. The targeting and therapeutic moieties arelinked via an intracellularly cleavable linkage that increasestherapeutic efficacy.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer, and virtually noapplication in other diseases, such as infectious and autoimmunediseases. The toxic agent is most commonly a chemotherapy drug, althoughparticle-emitting radionuclides, or bacterial or plant toxins have alsobeen conjugated to MAbs, especially for the therapy of cancer (Sharkeyand Goldenberg, CA Cancer J. Clin. 2006 July-August; 56(4):226-243) and,more recently, with radioimmunoconjugates for the preclinical therapy ofcertain infectious diseases (Dadachova and Casadevall, Q J Nucl Med MolImaging 2006; 50(3):193-204; incorporated herein by reference in itsentirety).

The advantages of using MAb-chemotherapy drug conjugates are that (a)the chemotherapy drug itself is structurally well defined; (b) thechemotherapy drug is linked to the MAb protein using very well definedconjugation chemistries, often at specific sites remote from the MAbsantigen binding regions; (c) MAb-chemotherapy drug conjugates can bemade more reproducibly than chemical conjugates involving MAbs andbacterial or plant toxins, and as such are more amenable to commercialdevelopment and regulatory approval; and (d) the MAb-chemotherapy drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates.

The present disclosure solves specific problems associated with thepreparation of conjugates of the camptothecin (CPT) group of cytotoxiccompounds. CPT and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad. Sci., NY922:1-10 (2000)).

CPTs present a set of caveats in the preparation of conjugates. Onecaveat is the insolubility of most CPT derivatives in aqueous buffers.Secondly, CPTs provide specific challenges for structural modificationfor conjugating to macromolecules. For instance, CPT itself containsonly a tertiary hydroxyl group in ring-E. The hydroxyl functional groupin the case of CPT must be coupled to a linker suitable for subsequentprotein conjugation; and in potent CPT derivatives, such as SN-38, theactive metabolite of the chemotherapeutic CPT-11, and otherC-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of phenolic hydroxyl at C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Thirdly thelability of the δ-lactone moiety of the E-ring of their structures,under physiological conditions, results in greatly reduced antitumorpotency of these products. Therefore, the conjugation protocol isperformed such that it is carried out at a pH of 7 or lower to avoid thelactone ring opening. Typically conjugation of a bifunctional CPTpossessing an amine-reactive group such as an active ester would requirea pH of 8 or greater. Fourth, an intracellularly-cleavable moiety is tobe incorporated in the linker/spacer connecting the CPTs and theantibodies or other binding moieties.

The problem of δ-lactone opening under physiological conditions has beenpreviously addressed. One approach has been to acylate the C-20 hydroxylgroup with an amino acid, and couple the α-amino group of the amino acidto poly-L-glutamic acid (Singer et al. in The Camptothecins: UnfoldingTheir Anticancer Potential, Liehr J. G., Giovanella, B. C. andVerschraegen (eds), NY Acad. Sci., NY 922:136-150 (2000)). This approachrelies on the passive diffusion of a polymeric molecule into tumorsites. This glycine conjugation has also been reported as a method ofmaking water-soluble derivative of CPT (Vishnuvajjala et al., U.S. Pat.No. 4,943,579) and in the preparation of a PEG-derivatization of CPT(Greenwald, et al. J. Med. Chem. 39: 1938-1940 (1996). In the lattercase, the approach has been devised in the context of developingwater-soluble and long acting forms of CPT, whereby CPT's in vivohalf-life is enhanced, and the drug is gradually released from itsconjugate while in circulation in vivo.

The present invention discloses methods for preparing conjugates ofCPTs, of 10-hydroxy derivatives such as SN-38 in particular, taking intoconsideration the four caveats described above and the syntheticchallenges. SN-38 is the active drug form of the approved cancer drugCPT-11, which is a prodrug. Vast clinical data are available concerningCPT-11 pharmacology and of its in vivo conversion to SN-38 (Iyer andRatain, supra; Mathijssen et al., Clin Cancer Res. 7:2182-2194 (2002);Rivory, Ann NY Acad. Sci. 922:205-215, 2000)). The active form SN-38 isabout 2 to 3 orders of magnitude more potent than CPT-11.

Early work on protein-drug conjugates indicated that a drug ideallyneeded to be released in its original form, once it had beeninternalized into a target cell, for the protein-chemotherapy drugconjugate to be a useful therapeutic. Trouet et al. (Proc. Natl. Acad.Sci. USA 79:626-629 (1982)) showed the advantage of using specificpeptide linkers, between the drug and the targeting moiety, which arecleaved lysosomally to liberate the intact drug. Work during the 1980'sand early 1990's focused further on the nature of the chemical linkerbetween the chemotherapeutic drug and the MAb. Notably, MAb-chemotherapydrug conjugates prepared using mild acid-cleavable linkers weredeveloped, based on the observation that the pH inside tumors was oftenlower than normal physiological pH. In this respect, superior resultswere found by incorporating a hydrazone as a cleavable unit, andattaching DOX to a MAb via a thioether group, (Willner et al., U.S. Pat.No. 5,708,146; Trail et al. (Science 261:212-215 (1993)).

This approach showed that MAb-doxorubicin (DOX) conjugates, preparedwith appropriate linkers, could be used to cure mice bearing a varietyof human tumor xenografts, in preclinical studies. The first approvedMAb-drug conjugate, Gemtuzumab Ozogamicin, incorporates a similaracid-labile hydrazone bond between an anti-CD33 antibody, humanizedP67.6, and a potent calicheamicin derivative. Sievers et al., J ClinOncol. 19:3244-3254 (2001); Hamann et al., Bioconjugate Chem. 13: 47-58(2002). In some cases, the MAb-chemotherapy drug conjugates were madewith reductively labile hindered disulfide bonds between thechemotherapy drugs and the MAb (Liu et al., Proc Natl Acad Sci USA 93:8618-8623 (1996)). Yet another cleavable linker involves a cathepsinB-labile dipeptide spacers, such as Phe-Lys or Val-Cit, similar to thelysosomally labile peptide spacers of Trouet et al. containing from oneto four amino acids, which additionally incorporated a collapsiblespacer between the drug and the dipeptide (Dubowchik, et al.,Bioconjugate Chem. 13:855-869 (2002); Firestone et al., U.S. Pat. No.6,214,345 B1; Doronina et al., Nat. Biotechnol. 21: 778-784 (2003)). Thelatter approaches were also utilized in the preparation of animmunoconjugate of camptothecin (Walker et al., Bioorg Med Chem. Lett.12:217-219 (2002)). Another cleavable moiety that has been explored isan ester linkage incorporated into the linker between the antibody andthe chemotherapy drug. Gillimard and Saragovi have found that when anester of paclitaxel was conjugated to anti-rat p75 MAb, MC192, oranti-human TrkA MAb, 5C3, the conjugate was found to exhibittarget-specific toxicity. Gillimard and Saragovi, Cancer Res. 61:694-699(2001).

While the importance of cleavable linker in the design of bindingmoiety-drug conjugates cannot be overstated, it is also important tofocus on how the linker design impacts the overall preparation ofspecific CPT-binding moiety conjugates. The present invention solves theproblem associated with the preparation of the bifunctional drug-linkermolecule, wherein the said drug may also contain more than one reactivegroup for derivatization, such as the potent SN-38 analog, for instance,in the design of conjugates. SN-38, a clinically important active drugform of the cancer drug CPT-11, but 100-1000-times more potent thanCPT-11, is not useable systemically because of insolubility. The presentinvention solves this problem by conjugating it to a targeting moiety inways that also address other challenges of using a CPT, whileconcurrently improving the therapeutic index of this clinicallyimportant potent drug by using disease-specific antibodies.

The conjugates of the instant invention possess greater efficacy, inmany cases, than unconjugated or “naked” antibodies or antibodyfragments, although such unconjugated targeting molecules have been ofuse in specific situations. In cancer, for example, naked antibodieshave come to play a role in the treatment of lymphomas (CAMPATH® andRITUXAN®), colorectal and other cancers (ERBITUX® and AVASTIN®), breastcancer (HERECEPTIN®), as well as a large number now in clinicaldevelopment (e.g., epratuzumab). In most of these cases, clinical usehas involved combining these naked, or unconjugated, antibodies withother therapies, such as chemotherapy or radiation therapy.

A variety of antibodies are also in use for the treatment of autoimmuneand other immune dysregulatory diseases, such as tumor necrosis factor(TNF) and B-cell (RITUXAN®) antibodies in arthritis, and are beinginvestigated in other such diseases, such as the B-cell antibodies,RITUXAN® and epratuzumab, in systemic lupus erythematosus and Sjögren'ssyndrome, as well as juvenile diabetes and multiple sclerosis. Nakedantibodies are also being studied in sepsis and septic shock,Alzheimer's disease, and infectious diseases. The development ofanti-infective monoclonal antibodies has been reviewed recently byReichert and Dewitz (Nat Rev Drug Discovery 2006; 5:191-195),incorporated herein by reference, which summarizes the prioritypathogens against which naked antibody therapy has been pursued,resulting in only 2 pathogens against which antibodies are either inPhase III clinical trials or are being marketed (respiratory syncytialvirus and methicillin-resistant Staphylococcus aureus), with 25 othersin clinical studies and 20 discontinued during clinical study.

Thus, there is a need to develop more potent anti-pathogen antibodiesand other binding moieties. Such antibody-mediated therapeutics can bedeveloped for the treatment of many different pathogens, includingbacteria, fungi, viruses, and parasites, either as naked (unconjugated),radiolabeled, or drug/toxin conjugates. In the case of deliveringdrug/toxin or radionuclide conjugates, this can be accomplished bydirect antibody conjugation or by indirect methods, referred to aspretargeting, where a bispecific antibody is used to target to thelesion, while the therapeutic agent is secondarily targeted by bindingto one of the arms of the bispecific antibody that has localized at thesite of the pathogen or of the cancer or whatever lesion is beingtreated (discussed by Goldenberg et al., J Clin Oncol. 2006 Feb. 10;24(5):823-34; and Goldenberg et al., J Nucl Med. 2008 January;49(1):158-63, both incorporated in their entirety herein by reference).

SUMMARY OF THE INVENTION

The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparation ofcamptothecin-binding moiety conjugates. The disclosed methods andcompositions are of use for the treatment of a variety of diseases andconditions which are refractory or less responsive to other forms oftherapy, and can include diseases against which suitable targeting(binding) moieties for selective targeting can be developed, or areavailable or known. Preferably, the targeting moiety is an antibody,antibody fragment, bispecific or other multivalent antibody, or otherantibody-based molecule or compound. The antibody can be of variousisotypes, preferably IgG1, IgG2a, IgG3, IgG4, and IgA, and can be achimeric human-mouse, a chimeric human-primate, a humanized (humanframework and murine hypervariable (CDR) regions), or fully human MAbs,as well as variations thereof, such as half-IgG4 antibodies, referred toas “Unibodies,” as described by van der Neut Kolfschoten et al. (Science2007; 317:1554-1557), incorporated herein by reference in its entirety.However, other binding moieties known in the art, such as aptamers,avimers or targeting peptides, may be used. Preferred diseases orconditions against which such targeting moieties exist are, for example,cancer, immune dysregulatory conditions, including autoimmune diseasesand inflammatory diseases, and diseases caused by infectious organisms.

The disclosed methods and compositions may thus be applied for treatmentof diseases and conditions for which targeting moieties are of use todeliver camptothecin-related cytotoxic agents. Such diseases orconditions may be characterized by the presence of a target molecule ortarget cell that is insufficiently affected when unconjugated, or naked,targeting moieties are used, such as in the immunotherapy of cancer orof infection with pathogenic organisms. (For methods of makingimmunoconjugates of antibodies with isotopes, drugs, and toxins for usein disease therapies, see, e.g., U.S. Pat. Nos. 4,699,784; 4,824,659;5,525,338; 5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284;6,306,393; 6,653,104; 6,962,702; and U.S. Patent Appln. Publ. Nos.20050191239; 20050175582; 20050136001; 20040166115; 20040043030;20040022725; 20030068322; 20030031669; 20030026764 and 20020136690, eachincorporated by reference in its entirety.)

In certain exemplary embodiments, camptothecin conjugates of antibodiesor antibody fragments may be used for targeting this therapeutic drug topathogens, such as bacteria, viruses, fungi, and parasites. In preferredembodiments, such drug-conjugated targeting moieties can be used incombination with other therapeutic modalities, such as anti-fungal,antibiotics and anti-viral drugs and/or naked antibodies,immunomodulators (e.g., interferon, interleukins and/or othercytokines). The use of radioimmunotherapy for the treatment ofinfectious organisms is disclosed, for example, in U.S. Pat. Nos.4,925,648; 5,332,567; 5,439,665; 5,601,825; 5,609,846; 5,612,016;6,120,768; 6,319,500; 6,458,933; 6,548,275; and in U.S. PatentApplication Publication Nos. 20020136690 and 20030103982, each of whichis incorporated herein by reference in its entirety.

In certain embodiments involving treatment of cancer, the camptothecinconjugates may be used in combination with surgery, radiation,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. Similar combinations arepreferred in the treatment of the other diseases amenable to targetingmoieties, such as autoimmune diseases. For example, the camptothecinconjugates can be combined with TNF inhibitors, B-cell antibodies,interferons, interleukins, and other effective agents for the treatmentof autoimmune diseases, such as rheumatoid arthritis, systemic lupuserythematosis, Sjögren's syndrome, multiple sclerosis, vasculitis, aswell as type-I diabetes (juvenile diabetes). These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. In viral diseases, these drugimmunoconjugates can be combined with other therapeutic drugs,immunomodulators, naked MAbs, or vaccines (e.g., MAbs against hepatitis,HIV, or papilloma viruses, or vaccines based on immunogens of theseviruses). Antibodies and antigen-based vaccines against these and otherviral pathogens are known in the art and, in some cases, already incommercial use.

In one embodiment, the invention relates to a process of preparingconjugates, wherein a CPT drug is first derivatized with a linker, whichsaid linker contains a reactive moiety that is capable of combining witha second linker that additionally contains a targeting-moiety-couplinggroup; wherein the first linker also possesses a defined polyethyleneglycol (PEG) moiety for water-solubility, and optionally anintracellularly-cleavable moiety cleavable by intracellular peptidasesor cleavable by the low pH environment of endosomal and lysosomalvesicles, and optionally an amino acid spacer between the said drug andthe first linker; wherein the second linker contains a reactive groupcapable of reacting with drug-(first linker) conjugate by the copper(+1) ion-catalyzed acetylene-azide cycloaddition reaction, referred toas ‘click chemistry’ in the art.

In another embodiment, the invention relates to a process of preparingconjugates as given in the paragraph above, wherein the second linkerhas a single targeting-moiety-coupling group, but multiples of thereactive group capable of reacting with drug-(first linker) conjugate,thereby amplifying the number of drug molecules conjugated to thetargeting moiety.

In a further embodiment, the invention relates to a process of preparingconjugates,

wherein the linker is first conjugated to a CPT drug, thereby producinga CPT drug-linker conjugate; wherein said CPT drug-linker conjugatepreparation involves the selective protection and deprotection of C-10hydroxyl group, keeping the C-20 carbonate bond essentially intact, inderivatives of CPT containing a C-10 hydroxyl group; wherein saiddrug-linker conjugate is optionally not purified; and wherein saiddrug-linker conjugate is subsequently conjugated to a monoclonalantibody or fragment.

Yet another embodiment of the invention is a method of treating cancer(a malignancy), an autoimmune disease, an infection, or an infectiouslesion with the conjugates described herein. Alternative embodimentsconcern the drug-targeting moiety conjugates made by the claimedprocesses and/or kits for performing the claimed processes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of particularembodiments of the invention. The embodiments may be better understoodby reference to one or more of these drawings in combination with thedetailed description presented herein.

FIG. 1. Hydrolytic stability of hMN-14-[SN38-CL‘x’](‘x’=1, 2, 3, 4, 5)and hMN-14-[EtO-CO-10-O—SN38-CL2] conjugates in PBS, pH 7.4, 37° C.

FIG. 2. Cell binding of various hMN14-SN38 immunoconjugates on a humancolorectal adenocarcinoma cell line LoVo.

FIG. 3. In vitro cytotoxicity of various hMN14-SN38 immunoconjugates ona human colorectal adenocarcinoma cell line LoVo.

FIG. 4. Cytotoxicity against lung adenocarcinoma (Calu-3).

FIG. 5. Survival curves of hMN14-CL-SN38 treated mice bearing GW-39 lungmetastatic disease.

FIG. 6. Therapeutic efficacy of hPAM4-CL2-SN38 in CaPan1 tumor-bearingmice (n=4) (500 μg, q4dx8).

DEFINITIONS

Unless otherwise specified, “a” or “an” means “one or more.”

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention. Terms that are not expressly defined herein are usedin accordance with their plain and ordinary meanings.

The term targeting moiety as used herein refers to a molecule, complexor aggregate, that binds specifically or selectively to a targetmolecule, cell, particle, tissue or aggregate. In preferred embodiments,a targeting moiety is an antibody, antibody fragment, bispecificantibody or other antibody-based molecule or compound. However, otherexamples of targeting moieties are known in the art and may be used,such as aptamers, avimers, receptor-binding ligands, nucleic acids,biotin-avidin binding pairs, binding peptides or proteins, etc. Theterms “targeting moiety” and “binding moiety” are used synonymouslyherein.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. Anantibody or antibody fragment may be conjugated or otherwise derivatizedwithin the scope of the claimed subject matter. Such antibodies includeIgG1, IgG2a, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv) and the like, includingthe half-molecules of IgG4 cited above (van der Neut Kolfschoten et al.(Science 2007; 317(14 September):1554-1557). Regardless of structure, anantibody fragment of use binds with the same antigen that is recognizedby the intact antibody. The term “antibody fragment” also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments consisting of the variableregions, such as the “Fv” fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“scFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion, such as CDRs. The Fv fragments may be constructed in differentways to yield multivalent and/or multispecific binding forms. In theformer case of multivalent, they react with more than one binding siteagainst the specific epitope, whereas with multispecific forms, morethan one epitope (either of the same antigen or against the specificantigen and a different antigen) is bound. As used herein, the termantibody component includes an entire antibody, a fusion protein, andfragments thereof.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector or immunological functions, such ascomplement fixation and ADCC (antibody-dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,the Fc portion may not be required for therapeutic function of theantibody, but rather other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,such as inhibition of heterotypic or homotypic adhesion, andinterference in signaling pathways, may come into play and interferewith the disease progression. Naked antibodies include both polyclonaland monoclonal antibodies, and fragments thereof, that include murineantibodies, as well as certain recombinant antibodies, such as chimeric,humanized or human antibodies and fragments thereof. Therefore, in somecases a “naked antibody” may also refer to a “naked” antibody fragment.As defined herein, “naked” is synonymous with “unconjugated,” and meansnot linked or conjugated to a therapeutic agent with which itadministered.

Autoimmune Diseases are disorders that are caused by the body producingan immune response against its own tissues. Examples include Class IIIautoimmune diseases such as immune-mediated thrombocytopenias, acuteidiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sjögren's syndrome, multiplesclerosis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, as disclosed in U.S.Provisional Application Ser. No. 60/360,259, filed Mar. 1, 2002,incorporated herein by reference in its entirety.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a subhuman primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule are derived from those of a human antibody. In some cases,specific residues of the framework region of the humanized antibody,particularly those that are touching or close to the CDR sequences, maybe modified, for example replaced with the corresponding residues fromthe original rodent, subhuman primate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain locus are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy chain and light chain loci. The transgenic micecan synthesize human antibodies specific for human antigens, and themice can be used to produce human antibody-secreting hybridomas. Methodsfor obtaining human antibodies from transgenic mice are described byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporatedherein by reference in their entirety.

Infectious Diseases as used herein are diseases involving infection bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi,viruses, parasites, or other microbial agents. Examples include humanimmunodeficiency virus (HIV) causing AIDS, Mycobacterium oftuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes,West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellaabortus, rabies virus, influenza virus, cytomegalovirus, herpes simplexvirus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reo virus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference in its entirety.

A therapeutic agent is a molecule or atom that is administeredseparately, concurrently or sequentially with a binding moiety, e.g., anantibody or antibody fragment, or a subfragment thereof, and is usefulin the treatment of a disease. Examples of therapeutic agents include,but are not limited to, antibodies, antibody fragments, conjugates,drugs, cytotoxic agents, proapopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radioisotopes orradionuclides, oligonucleotides, interference RNA, peptides,anti-angiogenic agents, chemotherapeutic agents, cyokines, chemokines,prodrugs, enzymes, binding proteins or peptides, conjugates orcombinations thereof.

A conjugate is an antibody component or other targeting moietyconjugated to a therapeutic agent. Suitable therapeutic agents aredescribed above.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which two or more ofthe same or different natural antibody, single-chain antibody orantibody fragment segments with the same or different specificities arelinked. A fusion protein comprises at least one specific binding site.Valency of the fusion protein indicates the total number of binding armsor sites the fusion protein has to antigen(s) or epitope(s); i.e.,monovalent, bivalent, trivalent or mutlivalent. The multivalency of theantibody fusion protein means that it can take advantage of multipleinteractions in binding to an antigen, thus increasing the avidity ofbinding to the antigen, or to different antigens. Specificity indicateshow many different types of antigen or epitope an antibody fusionprotein is able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one type of antigen or epitope. A monospecific,multivalent fusion protein has more than one binding site for the sameantigen or epitope. For example, a monospecific diabody is a fusionprotein with two binding sites reactive with the same antigen. Thefusion protein may comprise a multivalent or multispecific combinationof different antibody components or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise a therapeuticagent.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells, as in therapeutic treatment of autoimmune disease. An example ofan immunomodulator as described herein is a cytokine, which is a solublesmall protein of approximately 5-20 kDa that is released by one cellpopulation (e.g., primed T-lymphocytes) on contact with specificantigens, and which acts as an intercellular mediator between cells. Asthe skilled artisan will understand, examples of cytokines includelymphokines, monokines, interleukins, and several related signalingmolecules, such as tumor necrosis factor (TNF) and interferons.Chemokines are a subset of cytokines. Certain interleukins andinterferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation.

CPT is abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin. The structures of camptothecin and some ofits analogs, with the numbering indicated and the rings labeled withletters A-E, are given in formula 1 in Chart 1 below.

DETAILED DESCRIPTION OF THE INVENTION

Methods are devised in the following ways for the preparation ofconjugates of CPT or a CPT analog or derivative (collectively ‘CPT’)with targeting moiety such as an antibody (MAb). The disclosed methodsrepresent a preferred embodiment of the invention. (1) Solubility of CPTis enhanced by placing a defined polyethylene glycol moiety (PEG)between CPT and the targeting vector; (2) a first linker connects thedrug at one end and terminates with an acetylene or an azide oracetylene group at the other end; this first linker comprises a definedPEG moiety with an azide or acetylene group at one end and a differentreactive group, such as carboxylic acid or hydroxy group, at the otherend and said bifunctional defined PEG is attached to a CPT-20-esterderived from an amino acid or to the CPT-20-O-chloroformate;alternatively, the non-azide (or acetylene) moiety of said definedbifunctional PEG is optionally attached to a cleavable linker, cleavableby intracellular peptidases or by low pH in certain intracellularcompartments, and the cleavable linker is attached to the drug; (3) asecond linker, comprising a targeting moiety-coupling group and areactive group complementary to the azide (or acetylene) group of thefirst linker, namely acetylene (or azide), reacts with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnishthe final bifunctional drug product that is useful for conjugating tothe disease targeting moieties such as disease-targeting antibodies; (4)the antibody-coupling group is designed to be either a thiol or athiol-reactive group; (5) methods are devised for selective regenerationof the 10-hydroxyl group in presence of the C-20 carbonate inpreparations of drug-linker precursor involving CPT analogs such asSN-38; and (6) the 10-hydroxyl group of CPT analogs is alternativelyprotected as an ester or carbonate, other than ‘BOC’, such that thebifunctional CPT is conjugated to targeting moiety without priordeprotection of this protecting group, and the protecting group isreadily deprotected under the physiological pH condition after thebioconjugate is administered. In the acetylene-azide coupling, referredto as ‘click chemistry’ in the art, the azide part may be on L2 withacetylene part on L3; alternatively, L2 may contain acetylene, with L3containing azide. ‘Click chemistry’ is a copper (+1)-catalyzedcycloaddition reaction between an acetylene moiety and an azide moiety,and is a relatively recent technique in bioconjugations (Kolb H C andSharpless K B, Drug Discov Today 2003; 8: 1128-37). Click chemistrytakes place in aqueous solution at near-neutral pH conditions, and isthus amenable for drug conjugation. The advantage of click chemistry isthat it is chemoselective, and complements other well-known conjugationchemistries such as the thiol-maleimide reaction. In the followingdiscussion, where a conjugate comprises an antibody or antibodyfragment, another type of binding moiety, such as an aptamer, avimer ortargeting peptide, may be substituted.

An exemplary preferred embodiment is directed to a conjugate of acamptothecin drug derivative and an antibody of the general formula 2,

MAb-[L3]-[L2]-[L1]_(m)AA_(n)-CPT  (2)

where MAb is a disease-targeting antibody; CPT is camptothecin (CPT) oran analog thereof; L-3 is a component of the cross-linker comprising anantibody-coupling moiety and one or more of acetylene (or azide) groups;L2 comprises a defined PEG with azide (or acetylene) at one end,complementary to the acetylene (or azide) moiety in linker 3, and areactive group such as carboxylic acid or hydroxyl group at the otherend; L1 comprises a collapsible linker, or a peptidase-cleavable moietyoptionally attached to a collapsible linker, or an acid-cleavablemoiety; AA is an amino acid; m is an integer with values of 0 or 1, andn is an integer with values of 0, 1, 2, 3, or 4.

In a preferred embodiment, m is 0. In this embodiment, an ester moietyis first formed between the carboxylic acid of an amino acid such asglycine, alanine, or sarcosine, or of a peptide such as glycylglycine,and the 20-hydroxyl of CPT. In these cases, the N-terminus of the aminoacid or polypeptide is protected as a Boc or a Fmoc or amonomethoxytrityl (MMT) derivative, which is deprotected after formationof an ester bond with the 20-hydroxyl of CPT. Selective removal ofamine-protecting group, in presence of a BOC protecting group at theC-10-hydroxyl position of CPT analogs containing the additional10-hydroxyl group, as in some analogs shown in Chart 1, is achievedusing monomethoxytrityl (MMT) as the protecting group for the aminogroup of amino acid or polypeptide involved in ester formation, since‘MMT’ is removable by mild acid treatment such as dichloroacetic acidthat does not cleave a BOC group. After the amino group of the aminoacid or polypeptide, forming an ester bond with CPT at the 20 position,is demasked, the amino group is reacted with the activated form of COOHgroup on PEG moiety of L1 under standard amide-forming conditions. In apreferred embodiment, L3 comprises a thiol-reactive group which links tothiol groups of said targeting moiety. The thiol-reactive group isoptionally a maleimide or vinylsulfone, or bromoacetamide, oriodoacetamide, which links to thiol groups of said targeting moiety. Ina preferred embodiment, said reagent bearing a thiol-reactive group isgenerated from succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SMCC) or fromsuccinimidyl-ε-maleimido)caproate, for instance, with the thiol-reactivegroup being a maleimide group.

In a preferred embodiment, m is 0, and AA comprises polypeptide moiety,preferably tri or tetrapeptide, that is cleavable by intracellularpeptidase such as Cathepsin-B. Examples of cathepsin-B-cleavablepeptides are: Phe-Lys, Val-Cit (Dubowchick, 2002), Ala-Leu, Leu-Ala-Leu,and Ala-Leu-Ala-Leu (Trouet et al., 1982).

In a preferred embodiment, L1 is composed of intracellularly-cleavablepolypeptide, such as cathepsin-B-cleavable peptide, connected to thecollapsible linker p-aminobenzyl alcohol at the peptide's C-terminus,the benzyl alcohol portion of which is in turn directly attached toCPT-20-O-chloroformate. In this embodiment, n is 0. Alternatively, when‘n’ is non-zero, the benzyl alcohol portion of the p-amidobenzyl alcoholmoiety is attached to the N-terminus of the amino acid or polypeptidelinking at CPT's 20 position through the activated form of p-amidobenzylalcohol, namely PABOCOPNP where PNP is p-nitrophenyl. In a preferredembodiment, the linker comprises a thiol-reactive group which links tothiol groups of said targeting moiety. The thiol-reactive group isoptionally a maleimide or vinylsulfone, or bromoacetamide, oriodoacetamide, which links to thiol groups of said targeting moiety. Ina preferred embodiment, said reagent bearing a thiol-reactive group isgenerated from succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SMCC) or fromsuccinimidyl-(ε-maleimido)caproate, for instance, with thethiol-reactive group being a maleimide group.

In a preferred embodiment, the L2 component of the conjugate contains apolyethylene glycol (PEG) spacer that can be of up to MW 5000 in size,and in a more preferred embodiment, PEG is a defined PEG with (1-12 or1-30) repeating monomeric units. In a further preferred embodiment, PEGis a defined PEG with 1-12 repeating monomeric units. The introductionof PEG may involve using heterobifunctionalized PEG derivatives whichare available commercially. In the context of the present invention, theheterobifunctional PEG contains an azide or acetylene group. An exampleof a heterobifunctional defined PEG containing 8 repeating monomericunits, with ‘NHS’ being succinimidyl, is given below in formula 3:

In a preferred embodiment, L3 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single targeting vector-binding moiety.

A representative SN-38 (a CPT analog) conjugate of an antibody, preparedwith a maleimide-containing SN-38-linker derivative, with the bonding toMAb represented as a succinimide, is given below. Here, m=0, and the20-O-AA ester bonding to SN-38 is glycinate; azide-acetylene couplingjoining the L2 and L3 parts of formula 2 results in the triazole moietyas shown.

A representative SN-38 conjugate of an antibody, prepared with amaleimide-containing SN-38-linker derivative, with the bonding to MAbrepresented as a succinimide, is given below. Here, n=0 in the generalformula 2; ‘L1’ contains a cathepsin-B-cleavable dipeptide attached tothe collapsible p-aminobenzyl alcohol moiety, and the latter is attachedto SN-38 as a carbonate bonding at the 20 position; azide-acetylenecoupling joining the ‘L2’ and ‘L3’ parts of formula 2 results in thetriazole moiety as shown.

A representative SN-38 conjugate of an antibody, prepared with amaleimide-containing SN-38-linker derivative, with the bonding to MAbrepresented as a succinimide, is given below. Here, the 20-O-AA esterbonding to SN-38 is glycinate that is attached to L1 portion via ap-aminobenzyl alcohol moiety and a cathepsin-B-cleavable dipeptide; thelatter is in turn attached to ‘L2’ via an amide bond, while ‘L2’ and‘L3’ parts of general formula 2 are coupled via azide-acetylene ‘clickchemistry’ as shown.

A representative SN-38 conjugate of an antibody, prepared with amaleimide-containing SN-38-linker derivative, with the bonding to MAbrepresented as a succinimide, is given below. Here, n=0 in the generalformula 2; ‘L1’ contains just the collapsible p-aminobenzyl alcoholmoiety, and the latter is attached to SN-38 as a carbonate bonding atthe 20 position; azide-acetylene coupling joining the ‘L2’ and ‘L3’parts of formula 2 results in the triazole moiety as shown.

A representative SN-38 conjugate of an antibody, prepared with amaleimide-containing SN-38-linker derivative, with the bonding to MAbrepresented as a succinimide, is given below. Here, m=0, n=0 in thegeneral formula 2; ‘L2’ containing azido PEG is attached to SN-38 as acarbonate bonding at the 20 position; azide-acetylene coupling joiningthe ‘L2’ and ‘L3’ parts of formula 2 results in the triazole moiety asshown.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single targeting vector-binding moiety is shown below.‘L3’ component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Here,m=0, and the 20-O-AA ester bonding to SN-38 is glycinate;azide-acetylene coupling joining the L2 and L3 parts results inbis-triazole moiety as shown. The bonding to MAb is represented as asuccinimide.

A representative SN-38 conjugate of an antibody, prepared with amaleimide-containing SN-38-linker derivative, with the bonding to MAbrepresented as a succinimide, is given below. Here, the ‘AA’ isglycinate (i.e. SN-38-20-O-glycinate), which is reacted with anactivated acetoacetate. In the L1 portion, the PEG acid is converted tothe corresponding hydrazide and attached toSN38-20-O-glycinatoacetoacetate in the form of hydrazone.Azide-acetylene coupling joining the L1 and L2 parts results in thetriazole moiety as shown. The cleavable linker (‘CL’) is the hydrazonemoiety in the conjugate, which is cleavable by low pH conditions ofcertain intracellular compartments.

In preferred embodiments, when the bifunctional drug containsthiol-reactive moiety as antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple CPT moieties upon reaction with[L]-ester-CPT of the general formula given above, in the interchainregion of MAb away from antigen-binding region. By this way, the CPTwith a thiol-reactive group can be conjugated to MAb eithersite-specifically on the cysteines generated by disulfide reduction orindirectly on the lysine side chains of MAb derivatized to possess thiolgroups.

In a preferred embodiment of the present invention, the preferredchemotherapeutic moiety is selected from the group consisting of CPT,10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin, and derivatives thereof. In amore preferred embodiment, the chemotherapeutic moiety is SN-38.Preferably, in the conjugates of the preferred embodiments of thepresent invention, the targeting moiety links to at least onechemotherapeutic moiety; preferably 1 to about 12 chemotherapeuticmoieties; most preferably about 6 to about 12 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component ‘L3’comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidtargeting moiety. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of these embodiments, a process was surprisinglydiscovered by which CPT drug-linkers can be prepared wherein CPTadditionally has a 10-hydroxyl group. This process involves, but is notlimited to, the protection of the said 10-hydroxyl group as at-butyloxycarbonyl (BOC) derivative, followed by the preparation of thepenultimate intermediate of the drug-linker conjugate. Usually, removalof BOC group requires treatment with strong acid such as trifluoroaceticacid (TFA). Under these conditions, the CPT 20-O-linker carbonate,containing protecting groups to be removed, is also susceptible tocleavage, thereby giving rise to unmodified CPT. In fact, the rationalefor using a mildly removable methoxytrityl (MMT) protecting group forthe lysine side chain of the linker molecule, as enunciated in the art,was precisely to avoid this possibility (Walker et al., 2002). It wasdiscovered that selective removal of phenolic BOC protecting group ispossible by carrying out reactions for short durations, optimally 3-to-5minutes. Under these conditions, the predominant product was that inwhich the ‘BOC’ at 10-hydroxyl position was removed, while the carbonateat ‘20’ position was intact.

An alternative approach involves protecting CPT analogs' 10-hydroxyposition with a group other than ‘BOC’, such that the final product isready for conjugation to antibodies without a need for deprotecting the10-OH protecting group. The said 10-hydroxy protecting group, whichconverts the 10-OH into a phenolic carbonate or a phenolic ester, isreadily deprotected by physiological pH conditions or by esterases afterin vivo administration of the conjugates. The faster removal of aphenolic carbonate at 10 position vs. tertiary carbonate at 20 positionof 10-hydroxycamptothecin under physiological condition has beendescribed by He et al. (He et al., Bioorganic & Medicinal Chemistry 12:4003-4008 (2004)). Structure 11 below shows the SN-38 conjugate of thisembodiment, with a 10-hydroxy protecting group on SN-38 shown as ‘COR’where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—” where nis 2-10 and wherein the terminal amino group is optionally in the formof a quaternary salt for enhanced aqueous solubility, or simple alkylresidue such as “CH₃—(CH₂)_(n)—” where n is 2-10, or it can be alkoxymoiety such as “CH₃—(CH₂)_(n)—O—” or “N(CH₃)₂—(CH₂)_(n)—O—” where n is2-10, or “R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyland n is an integer with values of 0-10. These 10-hydroxy derivativesare readily prepared by treatment with the chloroformate of the chosenreagent, if the final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In one embodiment, the targeting moiety is a monoclonal antibody (MAb).In a further embodiment, the targeting moiety may be a multivalentand/or multispecific MAb. The targeting moiety may be a murine,chimeric, humanized, or human monoclonal antibody, and said antibody maybe in intact, fragment (Fab, Fab′, F(ab)₂, F(ab′)₂), or sub-fragment(single-chain constructs) form, or of an IgG1, IgG2a, IgG3, IgG4, IgAisotype, or submolecules therefrom.

In a preferred embodiment, the targeting moiety is a monoclonal antibodythat is reactive with an antigen or epitope of an antigen expressed on acancer or malignant cell. The cancer cell is preferably a cell from ahematopoietic tumor, carcinoma, sarcoma, melanoma or a glial tumor. Apreferred malignancy to be treated according to the present invention isa malignant solid tumor or hematopoietic neoplasm.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof, and particularly cleaved byesterases and peptidases.

The targeting moiety is preferably an antibody (including fully human,non-human, humanized, or chimeric antibodies) or an antibody fragment(including enzymatically or recombinantly produced fragments) or bindingproteins incorporating sequences from antibodies or antibody fragments.The antibodies, fragments, and binding proteins may be multivalent andmultispecific or multivalent and monospecific as defined above.

Multivalent and multispecific or multivalent and monospecific bindingproteins may be fusion proteins. In a preferred embodiment, the fusionproteins are assembled by the ‘dock and lock (DNL)’ technology (Rossi EA, et al., Proc Natl Acad Sci USA 2006; 103:6841-6846; U.S. PatentApplication Publication Nos. 20060228300; 20070086942 and 20070140966,the text of each of which is incorporated herein by reference in itsentirety). The DNL technique is based upon the formation of complexes ofnaturally occurring binding molecules, for example between thedimerization and docking domain (DDD) regions of the regulatory subunitsof cAMP-dependent protein kinase and the anchoring domain (AD) sequenceobtained from a wide variety of A-kinase anchoring proteins (AKAPs). TheDDD domains spontaneously dimerize and then bind to a single ADsequence. Thus, various effectors may be attached to DDD and ADsequences to form complexes of defined stoichiometry. In the simplestcase, the result is a trimer comprising two identical subunits thatincorporate a DDD sequence and one subunit that incorporates an ADsequence. However, many variations on such assemblages are possible,including homodimers, homotetramers, heterotetramers and homo orheterohexamers (see US Patent Application Publ. Nos. 20060228357 and20070140966). Exemplary DDD and AD sequences that may be utilized in theDNL method to form synthetic complexes are disclosed below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1 (SEQ IDNO: 3) QIEYLAKQIVDNAIQQ AD2 (SEQ ID NO: 4) CGQIEYLAKQIVDNAIQQAGC

In a preferred embodiment of the present invention, antibodies, such asMAbs, are used that recognize or bind to markers or tumor-associatedantigens that are expressed at high levels on target cells and that areexpressed predominantly or only on diseased cells versus normal tissues,and antibodies that internalize rapidly. Antibodies useful within thescope of the present invention include MAbs with properties as describedabove (and show distinguishing properties of different levels ofinternalization into cells and microorganisms), and contemplate the useof, but are not limited to, in cancer, the following MAbs: LL1(anti-CD74), LL2 and RFB4 (anti-CD22), RS7 (anti-epithelialglycoprotein-1 (EGP-1)), PAM-4 and KC4 (both anti-mucin), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e), MN-3 or MN-15(NCA or CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 (anti-PSMA(prostate-specific membrane antigen)), G250 (an anti-carbonic anhydraseIX MAb) and L243 (anti-HLA-DR). Such antibodies are known in the art(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;7,300,655; 7,312,318; and U.S. Patent Application Publ. No. 20040185053;20040202666; 20050271671; 20060193865; 20060210475; 20070087001; eachincorporated herein by reference in its entirety.)

Other useful antigens that may be targeted using these conjugatesinclude HER-2/neu, BrE3, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21,CD23, CD37, CD38, CD40, CD45, CD74, CD79, CD80, CD138, alpha-fetoprotein(AFP), VEGF (e.g. AVASTIN®, fibronectin splice variant), ED-Bfibronectin (e.g., L19), EGF receptor or ErbB1 (e.g., ERBITUX®), ErbB2,ErbB3, placental growth factor (P1GF), MUC1, MUC2, MUC3, MUC4, MUC5,PSMA, gangliosides, HCG, EGP-2 (e.g., 17-1A), CD37, HLA-DR, CD30, Ia,A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100, PSA (prostate-specificantigen), tenascin, folate receptor, Thomas-Friedenreich antigens, tumornecrosis antigens, tumor angiogenesis antigens, Ga 733, IL-2, IL-6,T101, MAGE, insulin-like growth factor (ILGF), macrophagemigration-inhibition factor (MIF), the HLA-DR antigen to which L243binds, CD66 antigens, i.e., CD66a-d or a combination thereof, as well ascancer stem cell antigens, such as CD133 and CD44.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623), all incorporated in their entirety by reference. Anumber of the aforementioned antigens are disclosed in U.S. ProvisionalApplication Ser. No. 60/426,379, entitled “Use of Multi-specific,Non-covalent Complexes for Targeted Delivery of Therapeutics,” filedNov. 15, 2002, incorporated herein by reference. Cancer stem cells,which are ascribed to be more therapy-resistant precursor malignantcells populations (Gan, J Cell Mol. Med. 2007 Dec. 5 [Epub ahead ofprint]; Hill and Perris, J. Natl. Cancer Inst. 2007; 99(19:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

In multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2007; October 9 (epubahead of print), and CD40 (Tai et al., 2005; Cancer Res.65(13):5898-5906).

A recent comprehensive analysis of suitable antigen (ClusterDesignation, or CD) targets on hematopoietic malignant cells, as shownby flow cytometry and which can be a guide to selecting suitableantibodies for drug-conjugated immunotherapy, is Craig and Foon, Bloodprepublished online Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535,incorporated herein in its entirety by reference.

In another preferred embodiment of the present invention, antibodies areused that, internalize rapidly and are then re-expressed, processed andpresented on cell surfaces, enabling continual uptake and accretion ofcirculating conjugate by the cell. An example of a most-preferredantibody/antigen pair is LL 1, an anti-CD74 MAb (invariant chain, classII-specific chaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104;7,312,318; and U.S. Patent Appl. Publ. Nos. 20020187153; 20030220470;20040185053; 20040202666; 20040219203; 20050271671; 20060014245;20060193865; 20070207146; each incorporated herein by reference in itsentirety). The CD74 antigen is highly expressed on B-cell lymphomas(including multiple myeloma) and leukemias, certain T-cell lymphomas,melanomas, colonic, lung, and renal cancers, glioblastomas, and certainother cancers (Ong et al., Immunology 98:296-302 (1999)), as well ascertain autoimmune diseases. A review of the use of CD74 antibodies incancer is contained in Stein et al., Clin Cancer Res. 2007 Sep. 15;13(18 Pt 2):5556s-5563s, incorporated herein by reference in itsentirety.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL 1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofa therapeutic conjugate as described herein to a subject. Diseases thatmay be treated with the therapeutic conjugates of the preferredembodiments of the present invention include, but are not limited toB-cell malignancies (e.g., non-Hodgkin's lymphoma and chroniclymphocytic leukemia using, for example LL2 MAb; see U.S. Pat. No.6,183,744), adenocarcinomas of endodermally-derived digestive systemepithelia, cancers such as breast cancer and non-small cell lung cancer,and other carcinomas, sarcomas, glial tumors, myeloid leukemias, etc. Inparticular, antibodies against an antigen, e.g., an oncofetal antigen,produced by or associated with a malignant solid tumor or hematopoieticneoplasm, e.g., a gastrointestinal, lung, breast, prostate, ovarian,testicular, brain or lymphatic tumor, a sarcoma or a melanoma, areadvantageously used. Such therapeutics can be given once or repeatedly,depending on the disease state and tolerability of the conjugate, andcan also be used optimally in combination with other therapeuticmodalities, such as surgery, external radiation, radioimmunotherapy,immunotherapy, chemotherapy, antisense therapy, interference RNAtherapy, gene therapy, and the like. Each combination will be adapted tothe tumor type, stage, patient condition and prior therapy, and otherfactors considered by the managing physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. The term subject also includes rodents (e.g., mice,rats, and guinea pigs). It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term.

In another preferred embodiment, the therapeutic conjugates comprisingthe Mu-9 MAb of the preferred embodiments of the present invention canbe used to treat colorectal, as well as pancreatic and ovarian cancersas disclosed in U.S. Pat. No. 6,962,702 and U.S. application Ser. No.10/116,116, filed Apr. 5, 2002, each incorporated herein by reference inits entirety and by Gold et al. (Cancer Res. 50: 6405 (1990), andreferences cited therein). In addition, the therapeutic conjugatescomprising the PAM-4 MAb of the preferred embodiments of the presentinvention can be used to treat pancreatic cancer, as disclosed in U.S.Provisional Application Ser. No. 60/388,314, filed Jun. 14, 2002, andU.S. Pat. Nos. 7,238,786 and 7,282,567 each incorporated herein byreference in its entirety.

In another preferred embodiment, the therapeutic conjugates comprisingthe RS7 MAb (binding to epithelial glycoprotein-1 [EGP-1] antigen) ofthe preferred embodiments can be used to treat carcinomas such ascarcinomas of the lung, stomach, urinary bladder, breast, ovary, uterus,and prostate, as disclosed in U.S. Provisional Application Ser. No.60/360,229, filed Mar. 1, 2002, and U.S. Pat. No. 7,238,785,incorporated herein by reference in its entirety and by Stein et al.(Cancer Res. 50: 1330 (1990) and Antibody Immunoconj. Radiopharm. 4: 703(1991)).

In another preferred embodiment, the therapeutic conjugates comprisingthe anti-AFP MAb of the preferred embodiments can be used to treathepatocellular carcinoma, germ cell tumors, and other AFP-producingtumors using humanized, chimeric and human antibody forms, as disclosedin U.S. Provisional Application Ser. No. 60/399,707, filed Aug. 1, 2002,and U.S. Pat. No. 7,300,655, incorporated herein by reference in itsentirety.

In another preferred embodiment, the therapeutic conjugates comprisinganti-tenascin antibodies can be used to treat hematopoietic and solidtumors and conjugates comprising antibodies to tenascin can be used totreat solid tumors, preferably brain cancers like glioblastomas.

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy with this invention.

In another preferred embodiment, the therapeutic conjugates of thepreferred embodiments can be used against pathogens, since antibodiesagainst pathogens are known. For example, antibodies and antibodyfragments which specifically bind markers produced by or associated withinfectious lesions, including viral, bacterial, fungal and parasiticinfections, for example caused by pathogens such as bacteria,rickettsia, mycoplasma, protozoa, fungi, and viruses, and antigens andproducts associated with such microorganisms have been disclosed, interalia, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg U.S. Pat.Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709and 4,624,846, each incorporated herein by reference, and in Reichertand Dewitz, cited above. In a preferred embodiment, the pathogens areselected from the group consisting of HIV virus causing AIDS,Mycobacterium tuberculosis, Streptococcus agalactiae,methicillin-resistant Staphylococcus aureus, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae,Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpessimplex virus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reovirus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, West Nile virus, Plasmodiumfalciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli,Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei,Schistosoma mansoni, Schistosomajapanicum, Babesia bovis, Elmeriatenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis,Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata,Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M.hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivariumand M. pneumoniae, as disclosed in U.S. Pat. No. 6,440,416, incorporatedherein by reference.

In a more preferred embodiment, drug conjugates of the present inventioncomprising anti-gp120 and other such anti-HIV antibodies can be used astherapeutics for HIV in AIDS patients; and drug conjugates of antibodiesto Mycobacterium tuberculosis are suitable as therapeutics fordrug-refractive tuberculosis. Fusion proteins of anti-gp120 MAb (antiHIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examinedfor antiviral properties (Van Oigen et al., J Drug Target, 5:75-91,1998). Attempts at treating HIV infection in AIDS patients failed,possibly due to insufficient efficacy or unacceptable host toxicity. Thedrug conjugates of the present invention advantageously lack such toxicside effects of protein toxins, and are therefore advantageously used intreating HIV infection in AIDS patients. These drug conjugates can begiven alone or in combination with other antibiotics or therapeuticagents that are effective in such patients when given alone. Candidateanti-HIV antibodies include the anti-envelope antibody described byJohansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agens Chemother. 2006; 50(5): 1773-9, all incorporated herein in theirentirety by reference. A preferred targeting agent for HIV is variouscombinations of these antibodies in order to overcome resistance.

In another preferred embodiment, diseases that may be treated using thetherapeutic conjugates of the preferred embodiments of the presentinvention include, but are not limited to immune dysregulation diseaseand related autoimmune diseases, including Class III autoimmune diseasessuch as immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpura and chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sjögren's syndrome, multiple sclerosis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerativecolitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa,ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, and also juvenile diabetes,as disclosed in U.S. Provisional Application Ser. No. 60/360,259, filedMar. 1, 2002. Typical antibodies useful in these diseases include, butare not limited to, those reactive with HLA-DR antigens, B-cell andplasma-cell antigens (e.g., CD19, CD20, CD21, CD22, CD23, CD4, CD5, CD8,CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40,CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1, Ia, HM1.24,and HLA-DR), IL-6, IL-17. Since many of these autoimmune diseases areaffected by autoantibodies made by aberrant B-cell populations,depletion of these B-cells by therapeutic conjugates involving suchantibodies-therapeutic agent conjugates described herein is a preferredmethod of autoimmune disease therapy, especially when B-cell antibodiesare combined, in certain circumstances, with HLA-DR antibodies and/orT-cell antibodies (including those which target IL-2 as an antigen, suchas anti-TAC antibody). In a preferred embodiment, the anti-B-cell,anti-T-cell, or anti-macrophage or other such antibodies of use in thetreatment of patients with autoimmune diseases also can be conjugated toresult in more effective therapeutics to control the host responsesinvolved in said autoimmune diseases, and can be given alone or incombination with other therapeutic agents, such as TNF inhibitors or TNFantibodies, unconjugated B- or T-cell antibodies, and the like.

In a preferred embodiment of this invention, a more effectiveincorporation into cells and pathogens can be accomplished by usingmultivalent, multispecific or multivalent, monospecific antibodies.Multivalent means the use of several binding arms against the same ordifferent antigen or epitope expressed on the cells, whereasmultispecific antibodies involve the use of multiple binding arms totarget at least two different antigens or epitopes contained on thetargeted cell or pathogen. Examples of such bivalent and bispecificantibodies are found in U.S. patent applications 60/399,707, filed Aug.1, 2002; 60/360,229, filed Mar. 1, 2002; 60/388,314, filed Jun. 14,2002; and 10/116,116, filed Apr. 5, 2002, each of which is incorporatedherein by reference in its entirety. These multivalent or multispecificantibodies are particularly preferred in the targeting of cancers andinfectious organisms (pathogens), which express multiple antigen targetsand even multiple epitopes of the same antigen target, but which oftenevade antibody targeting and sufficient binding for immunotherapybecause of insufficient expression or availability of a single antigentarget on the cell or pathogen. By targeting multiple antigens orepitopes, said antibodies show a higher binding and residence time onthe target, thus affording a higher saturation with the drug beingtargeted in this invention.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes, such as ²¹²Bi, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, 68Ga,⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc or ¹¹¹In. Non-radioactive metals,such as manganese, iron and gadolinium, are useful for nuclear imagingor MRI, when used along with the stably tethered structures and carriersdescribed herein, or as direct therapeutics (e.g., when a beta- alpha-or Auger-emitting radionuclude is used), all of which are contemplatedas useful herein. Macrocyclic chelates such as NOTA, DOTA, and TETA areof use with a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates, such asmacrocyclic polyethers for complexing ²²³ Ra, may be used. Therapeuticagents for use in combination with the camptothecin conjugate of thisinvention also include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and proapoptotic agents, particularlydoxorubicin, methotrexate, taxol, other camptothecins, and others fromthese and other classes of anticancer agents, and the like. Other cancerchemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purineanalogs, platinum coordination complexes, hormones, and the like.Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

Another class of therapeutic agents consists of radionuclides that emitα-particles (such as ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac),β-particles (such as ³²P, ³³P, ⁴⁷SC, ⁶⁷CU, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²PR, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, orAuger electrons (such as ¹¹¹In, ¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt,^(195m)Hg). Alternatively therapeutic agents may comprise a radioisotopeuseful for diagnostic imaging. Suitable radioisotopes may include thosein the energy range of 60 to 4,000 KeV, or more specifically, ¹⁸F, ⁵²Fe,⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)TC, ⁴⁵Ti,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and thelike, or a combination thereof. See, e.g., U.S. patent applicationentitled “Labeling Targeting Agents with Gallium-68” (Griffiths, G. L.and W. J. McBride, W. J, U.S. Provisional Application No. 60/342,104)which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga, ^(94m)Tc, and thelike, for imaging purposes; incorporated entirely by reference.Detection can be achieved, for example, by single photon emissioncomputed tomography (SPECT), or positron emission tomography (PET). Theapplication also may be for intraoperative diagnosis to identify occultneoplastic tumors. Imaging therapeutic agents may include one or moreimage enhancing agents, which may include complexes of metals selectedfrom the group consisting of chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III).

In still other embodiments, a therapeutic agent may comprise one or moreradioactive isotopes useful for killing neoplastic or other rapidlydividing cells, which include β-emitters (such as ³²P, ³³P, ⁴⁷Sc, ⁶⁷Cu,⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re), Auger electron emitters (such as ¹¹¹In,¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt, ^(195m)Hg), α-emitters (such as²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac), or a combination thereof.

Therapeutic agents to be used in concert with the camptothecinconjugates also may be toxins conjugated to targeting moieties. Toxinsthat may be used in this regard include ricin, abrin, ribonuclease(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, C A Cancer J. Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499,which is incorporated herein by reference in its entirety.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens. Some ofthe more preferred target combinations include the following. This is alist of examples of preferred combinations, but is not intended to beexhaustive.

TABLE 1 Some Examples of multispecific antibodies. First target Secondtarget MIF A second proinflammatory effector cytokine, especiallyHMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP- 1A, or MIP-1B MIF Proinflammatoryeffector receptor, especially IL-6R IL-13R, and IL-15R MIF Coagulationfactor, especially TF or thrombin MIF Complement factor, especially C3,C5, C3a, or C5a MIF Complement regulatory protein, especially CD46,CD55, CD59, and mCRP MIF Cancer associated antigen or receptor HMGB-1 Asecond proinflammatory effector cytokine, especially MIF, TNF-α, IL-1,or IL-6 HMGB-1 Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP- 1A, or MIP-1B HMGB-1 Proinflammatory effector receptorespecially MCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Coagulation factor,especially TF or thrombin HMGB-1 Complement factor, especially C3, C5,C3a, or C5a HMGB-1 Complement regulatory protein, especially CD46, CD55,CD59, and mCRP HMGB-1 Cancer associated antigen or receptor TNF-α Asecond proinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α,IL-1, or IL-6 TNF-α Proinflammatory effector chemokine, especiallyMCP-1, RANTES, MIP- 1A, or MIP-1B TNF-α Proinflammatory effectorreceptor, especially IL-6R IL-13R, and IL-15R TNF-α Coagulation factor,especially TF or thrombin TNF-α Complement factor, especially C3, C5,C3a, or C5a TNF-α Complement regulatory protein, especially CD46, CD55,CD59, and mCRP TNF-α Cancer associated antigen or receptor LPSProinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 LPS Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP- 1A, or MIP-1B LPS Proinflammatory effector receptor,especially IL-6R IL-13R, and IL-15R LPS Coagulation factor, especiallyTF or thrombin LPS Complement factor, especially C3, C5, C3a, or C5a LPSComplement regulatory protein, especially CD46, CD55, CD59, and mCRP TFor thrombin Proinflammatory effector cytokine, especially MIF, HMGB-1,TNF-α, IL-1, or IL-6 TF or thrombin Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP- 1A, or MIP-1B TF or thrombinProinflammatory effector receptor, especially IL-6R, IL-13R, and IL-15RTF or thrombin Complement factor, especially C3, C5, C3a, or C5a TF orthrombin Complement regulatory protein, especially CD46, CD55, CD59, andmCRP TF or thrombin Cancer associated antigen or receptor

Still other combinations, such as are preferred for cancer therapies,include CD20+CD22 antibodies, CD74+CD20 antibodies, CD74+CD22antibodies, CEACAM5 (CEA)+CEACAM6 (NCA) antibodies, insulin-like growthfactor (ILGF)+CEACAM5 antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies,CEACAM5+EGFR antibodies. Such antibodies need not only be used incombination, but can be combined as fusion proteins of various forms,such as IgG, Fab, scFv, and the like, as described in U.S. Pat. Nos.6,083,477; 6,183,744 and 6,962,702 and U.S. Patent ApplicationPublication Nos. 20030124058; 20030219433; 20040001825; 20040202666;20040219156; 20040219203; 20040235065; 20050002945; 20050014207;20050025709; 20050079184; 20050169926; 20050175582; 20050249738;20060014245 and 20060034759, each incorporated herein by reference intheir entirety.

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. Methods of construction and use of avimers are discussedin more detail below.

Production of Antibody Fragments

Methods of monoclonal antibody production are well known in the art andany such known method may be used to produce antibodies of use in theclaimed methods and compositions. Some embodiments of the claimedmethods and/or compositions may concern antibody fragments. Suchantibody fragments may be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments may be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment may befurther cleaved using a thiol reducing agent and, optionally, a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab fragmentsand an Fc fragment. Exemplary methods for producing antibody fragmentsare disclosed in U.S. Pat. No. 4,036,945; U.S. Pat. No. 4,331,647;Nisonoff et al., 1960, Arch. Biochem. Biophys., 89:230; Porter, 1959,Biochem. J., 73:119; Edelman et al., 1967, METHODS IN ENZYMOLOGY, page422 (Academic Press), and Coligan et al. (eds.), 1991, CURRENT PROTOCOLSIN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.)

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies areobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994).

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. A non-limiting example ofsuch a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol.Methods 231:11-23, incorporated herein by reference) from Abgenix(Fremont, Calif.). In the XenoMouse® and similar animals, the mouseantibody genes have been inactivated and replaced by functional humanantibody genes, while the remainder of the mouse immune system remainsintact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled to other molecules bychemical cross-linking or other known methodologies. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXenoMouse® system but may utilize any transgenic animal that has beengenetically engineered to produce human antibodies.

Avimers

In certain embodiments, the precursors, monomers and/or complexesdescribed herein may comprise one or more er sequences. Avimers are aclass of binding proteins somewhat similar to antibodies in theiraffinities and specifities for various target molecules. They weredeveloped from human extracellular receptor domains by in vitro exonshuffling and phage display. (Silverman et al., 2005, Nat. Biotechnol.23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220.) Theresulting multidomain proteins may comprise multiple independent bindingdomains, that may exhibit improved affinity (in some casessub-nanomolar) and specificity compared with single-epitope bindingproteins. (Id.) In various embodiments, avimers may be attached to, forexample, DDD and/or AD sequences for use in the claimed methods andcompositions. Additional details concerning methods of construction anduse of avimers are disclosed, for example, in U.S. Patent ApplicationPublication Nos. 20040175756, 20050048512, 20050053973, 20050089932 and20050221384, the Examples section of each of which is incorporatedherein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, each of which isincorporated herein by reference, disclose methods for preparing a phagelibrary. The phage display technique involves genetically manipulatingbacteriophage so that small peptides can be expressed on their surface(Smith and Scott, 1985, Science 228:1315-1317; Smith and Scott, 1993,Meth. Enzymol. 21:228-257).

The past decade has seen considerable progress in the construction ofphage-di splayed peptide libraries and in the development of screeningmethods in which the libraries are used to isolate peptide ligands. Forexample, the use of peptide libraries has made it possible tocharacterize interacting sites and receptor-ligand binding motifs withinmany proteins, such as antibodies involved in inflammatory reactions orintegrins that mediate cellular adherence. This method has also beenused to identify novel peptide ligands that may serve as leads to thedevelopment of peptidomimetic drugs or imaging agents (Arap et al.,1998a, Science 279:377-380). In addition to peptides, larger proteindomains such as single-chain antibodies may also be displayed on thesurface of phage particles (Arap et al., 1998a).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998a, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807, incorporated hereinby reference.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, eachincorporated herein by reference. Methods for preparation and screeningof aptamers that bind to particular targets of interest are well known,for example U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163, eachincorporated herein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the binding sequences of aptamers may be flankedby primer-binding sequences, facilitating the amplification of theaptamers by PCR or other amplification techniques.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S, Not all linkages in an oligomer need to beidentical.

Conjugation Protocols

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that are facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, rectal, transmucosal, intestinaladministration, intramuscular, subcutaneous, intramedullary,intrathecal, direct intraventricular, intravenous, intravitreal,intraperitoneal, intranasal, or intraocular injections. The preferredroutes of administration are parenteral. Alternatively, one mayadminister the compound in a local rather than systemic manner, forexample, via injection of the compound directly into a solid tumor.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

General

The intermediate Phe-Lys(MMT)-PABOH and the cross-linkersPhe-Lys(MMT)—PABOH, MC-Phe-Lys(MMT)-PABOH andMC-Phe-Lys(MMT)-PABOCOO-PNP, where MC is maleimidocaproyl, Phe isphenylalanine, Lys is lysine, MMT is monomethoxytrityl, PABOH isp-aminobenzyl alcohol, and PNP is p-nitrophenyl moiety, were synthesizedusing a published method (Dubowchik et al., 2002). CPT-20-O-acylderivatives of amino acids were prepared adapting a published method(Vishnuvajjala et al., U.S. Pat. No. 4,943,579). The azide precursors ofCL1-SN-38, CL2-SN-38, and CL3-SN-38 shown in Schemes 1-3 have beendescribed in the U.S. Patent Application corresponding to provisionalU.S. Patent Application Ser. No. 60/885,325, filed on Jan. 17, 2007, theentire text of which is incorporated herein by reference. Abbreviationsused below are: DCC, dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; PEG, polyethyleneglycol. Flash chromatography was done using 230-400 mesh silica gel andmethanol-dichloromethane gradient elution. Reverse phase HPLC wasperformed using a 7.8×300 mm C18 HPLC column, fitted with a precolumnfilter, and using the solvent gradient of 100% solvent A to 100% solventB in 10 minutes at a flow rate of 3 mL per minute and maintaining at100% solvent B at a flow rate of 4.5 mL per minute for 5 or 10 minutes.Solvent A was 0.3% aqueous ammonium acetate, pH 4.46 while solvent B was9:1 acetonitrile-aqueous ammonium acetate (0.3%), pH 4.46. HPLC wasmonitored by a dual in-line absorbance detector set at 360 nm and 254nm.

Example 1 Preparation of CL1-SN-38

CL1-SN-38 is represented in Scheme-1. The azide precursor of CL1-SN-38shown in the scheme has been described in the U.S. Patent Applicationcorresponding to provisional U.S. Patent Application Ser. No.60/885,325, filed on Jan. 17, 2007, the entire text of which isincorporated herein by reference. The reagent, namely4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide, wasprepared by reacting 0.107 g of SMCC and 0.021 mL of proparylamine(0.018 g; 1.01 equiv.) in dichloromethane using 1.1 equiv. ofdiisopropylethylamine. After 1 hr, the solvent was removed and theproduct was passed through a column of silica gel and eluted with 80:20mixture of ethylacetate-hexane to obtain 83 mg of the product (colorlesspowder). Electrospray mass spectrum showed peaks at m/e 275 (M+H) and abase peak at m/e 192 in the positive ion mode, consistent with thestructure calculated for C₁₅H₁₈N₂O₃: 275.1390 (M+H). found: 275.1394(exact mass). A solution of the azide precursor (0.208 g) in DMSO (0.5mL) was added to the acetylenic reagent (0.173 g; 3 equiv.), and moreDMSO (1.5 mL) was added, followed by 1 mL of water, 0.05 M aqueouscupric sulfate (0.21 mL; 0.05 equiv. w.r.t azide) and 0.5 M aqueoussodium ascorbate (0.21 mL, 0.5 equiv. w.r.t. azide). The somewhat cloudysolution was stirred at ambient temperature for 1 hr. Solvents wereremoved under high vacuum, and the residual gummy material was purifiedby flash chromatography using methanol-dichloromethane (0-8%) gradientelution. The product, CL1-SN-38, was obtained in 65% yield.Reverse-phase HPLC (method 1): ret. time 9.55 min. Electrospray massspectrum (positive ion mode) showed a peak at m/e 1283 (M+Na),consistent with the expected structure. Calculated for C₆₁H₈₁N₉O₂₀:1260.5670 (M+H) and 1282.5490 (M+Na). found 1260.5688 (M+H) and1282.5491 (M+Na).

Example 2 Preparation of CL2-SN-38

Synthesis is schematically shown in Scheme-2. The azide precursor ofCL2-SN-38 shown in the scheme has been described in the U.S. PatentApplication corresponding to provisional U.S. Patent Application Ser.No. 60/885,325, filed on Jan. 17, 2007, the entire text of which isincorporated herein by reference. The ‘click chemistry’ coupling of theazide precursor shown below with the acetylenic product described inExample 1 was carried out as follows. The azide (0.22 g, 0.127 mmol) andthe acetylenic reagent (0.105 g, 0.38 mmol) were mixed in 3 mL of DMSOand 0.8 mL of water. Solid cuprous bromide (0.036 g, 2 equiv.) wasadded, and the heterogeneous mixture was stirred for 10 min. More water(0.7 mL) was added, and the reaction was continued for 40 min. Solventswere removed, and the crude product was purified by flash chromatographyusing methanol-dichloromethane gradient (2-8%) elution. The product wasobtained in 47% yield. Reverse-phase HPLC (method 1): ret. time 13.35min. Electrospray mass spectrum (positive ion mode) showed a peak at m/e2022 (M+Na) consistent with the structure. Calculated forC₁₀₇H₁₃₀N₁₂O₂₆: 1999.9292 (M+H) and 2021.9111 (M+Na). found 1999.9292(M+H) and 2021.9114 (M+Na). The product was subjected to short-durationtreatment with deprotection cocktail (trifluoroacetic acid (TFA) 2 mL,dichloromethane 0.5 mL, anisole 0.12 mL, and water 0.06 mL).Purification of crude product, after removal of TFA and solvents, wascarried out by flash chromatography using methanol-dichloromethane (5%to 18%) gradient elution. The product, CL2-SN-38, had HPLC ret. time of10.06 min. Yield: 56%. Electrospray mass spectrum (positive ion mode)showed peaks at m/e 1628 (M+H) and 1650 (M+Na), consistent withstructure. Calculated for C₈₂H₁₀₆N₁₂O₂₃: 1627.7566 (M+H) and 1649.7386(M+Na). found 1627.7585 (M+H) and 1649.7400 (M+Na).

Example 3 Preparation of CL3-SN-38

The azide precursor of CL3-SN-38 (shown in scheme-3 below) has beendescribed in the U.S. Patent Application filed corresponding toprovisional U.S. Patent Application Ser. No. 60/885,325, filed on Jan.17, 2007, the entire text of which is incorporated herein by reference.The ‘click chemistry’ coupling of the azide precursor shown below withthe acetylenic product described in Example 1 was carried out asfollows. The azide (0.05 g, 0.03 mmol) and the acetylenic reagent (0.024g, 0.087 mmol) were mixed in 1 mL of DMSO and 1 mL of water. Solidcuprous bromide (0.0045 g, 1 equiv.) was added, and the heterogeneousmixture was stirred for 1 hr. The crude product was precipitated bydilution with water, and purified by flash chromatography usingmethanol-dichloromethane gradient (2-10%) elution. The product wasobtained in 76% yield. Reverse-phase HPLC (method 1): ret. time 11.65min. Electrospray mass spectrum (positive ion mode) showed peak at m/e1957 (M+H), consistent with structure. Calculated for C₁₀₄H₁₂₅N₁₃O₂₅:1956.8982 (M+H) and 1978.8801 (M+Na). found 1956.8926 (M+H) and1978.8711 (M+Na). The product was subjected to short-duration treatmentwith deprotection cocktail (trifluoroacetic acid (TFA) 2 mL,dichloromethane 0.5 mL, anisole 0.12 mL, and water 0.06 mL).Purification of crude product, after removal of TFA and solvents, wascarried out by precipitation in ethyl ether. The product, CL3-SN-38, hadHPLC ret. time of 9.81 min. Yield: 90%. Electrospray mass spectrum(positive ion mode) showed peaks at m/e 1685 (M+H) and 1707 (M+Na),consistent with structure. Calculated for C₈₄H₁₀₉N₁₃O₂₄: 1684.7781 (M+H)and 1706.7600 (M+Na). found 1684.7778 (M+H) and 1706.7611 (M+Na).

Example 4 Preparation of CL4-SN-38

Preparation is shown schematically in Scheme-4. The commerciallyavailableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol wasactivated with NHS, DCC, and catalytic amount of DMAP, and reacted withp-aminobezyl alcohol. The purified product was reacted with10-O-BOC-SN38-20-O-chloroformate in the manner described for CL2-SN-38preparation of Example 2. The purified product, obtained in 48% yield,had HPLC ret. time of 12.11 min. Electospray mass spectrum showed m/e at1179 (M+H). Calculated for C₅₇H₇₅N₇O₂₀: 1178.5139 (M+H) and 1200.4959(M+Na). found 1178.5138 (M+H) and 1200.4944 (M+Na). The azide precursor(0.14 g) and the acetylenic reagent (0.1 g, 3 equiv) were mixed in 3 mLof DMSO and 1 mL of water. Solid cuprous bromide (0.05 g, 3 equiv.) wasadded, and the heterogeneous mixture was stirred for 10 min. More water(0.5 mL) was added, and the reaction was continued for 30 min. Solventswere removed, and the crude product was purified by flash chromatographyusing methanol-dichloromethane gradient (0-8%) elution. The productobtained (0.135 g; 84% yield) had HPLC ret time of ret. time 11.5 min.Calculated for C₇₂H₉₃N₉O₂₃: 1452.6457 (M+H) and 1474.6276 (M+Na). found1452.6429 (M+H) and 1474.6262 (M+Na). The product was subjected toshort-duration treatment with deprotection cocktail as described inExample 2. Purification of crude product, after removal of TFA andsolvents, was carried out by flash chromatography usingmethanol-dichloromethan (1% to 8%) gradient elution. The product,CL4-SN-38, had HPLC ret. time of 10.31 min. Yield: 56%. Electrospraymass spectrum (positive ion mode) showed peaks at m/e 1353 (M+H),consistent with structure. Calculated for C₆₇H₈₅N₉O₂₁: 1352.5932 (M+H)and 1374.5752 (M+Na). found 1352.5907 (M+H) and 1374.5729 (M+Na).

Example 5 Preparation of CL5-SN-38

The azide precursor for this substrate was prepared in 3 steps fromSN-38, involving protection of 110-OH group as 10-O-BOC derivative,followed by 20-O-chloroformate formation and reaction withO-(2-azidoethyl) heptaethyleneglycol. The azido-SN-38 product waspurified by flash chromatography in the manner described in Example 2.The product, with HPLC ret. time: 12.4 min, also contained ˜8.5% ofunremoved starting material. The electrospray mass spectrum of thismaterial showed m/e at 915 (M+H). Calculated for C₄₄H₅₉N₅O₁₆: 914.4036(M+H). found 914.4034 (M+H). The click chemistry coupling of this azidoderivative with the acetylenic reagent of Example 1 was carried out with3 equivalents of the latter and 3 equivalents of cuprous bromide in amixture of DMSO and water (1:1 v/v), and purified by flashchromatography. Yield: 81%. HPLC ret time: 11.62 min. Electrospray massspectrum (positive ion mode) showed peaks at m/e 1189 (M+H), consistentwith structure. Calculated for C₅₉H₇₇N₇O₁₉: 1188.5354 (M+H). found1188.5323 (M+H). Deprotection using trifluoroacetic acid usingconditions described in Example 2, followed by flash chromatographicpurification yielded the title product (CL5-SN-38). HPLC: ret. time10.28 min. Electrospray mass spectrum (positive ion mode) showed peaksat m/e 1089 (M+H), consistent with structure. Calculated forC₅₄H₆₉N₇O₁₇: 1088.4822 (M+H) and 1110.4642 (M+Na). found 1088.4799 (M+H)and 1110.4632 (M+Na).

Example 6 Preparations of CL1-SN-38-10-O—COR, CL2-SN-38-10-O—COR,CL3-SN-38-10-O—COR, CL4-SN-38-10-O—COR, and CL5-SN-38-10-O—COR

This Example shows that the 10-OH group of SN-38 is protected as acarbonate or an ester, instead of as ‘BOC’, such that the final productis ready for conjugation to antibodies without a need for deprotectingthe 10-OH protecting group. This aspect has been described in paragraph053. This group is readily deprotected under physiological pH conditionsafter in vivo administration of the protein conjugate. In Scheme-6, ‘R’can be a substituted alkyl such as (CH₂)_(n)—N(CH₃)₂ where n is 2-10, orsimple alkyl such as (CH₂)_(n)—CH₃ where n is 2-10, or it can be alkoxymoiety such as “CH₃—(CH₂)_(n)—O—” or substituted alkoxy moiety such assuch as O—(CH₂)_(n)—N(CH₃)₂ where n is 2-10 and wherein the terminalamino group is optionally in the form of a quaternary salt for enhancedaqueous solubility, or it is a methoxy PEG residue. In the simplestversion of the latter category, R=“—O—(CH₂)₂—OCH₃”. These 10-hydroxyderivatives are readily prepared by treatment with the chloroformate ofthe chosen reagent, if the final derivative is to be a carbonate.Typically, the 10-hydroxy-containing camptothecin such as SN-38 istreated with a molar equivalent of the chloroformate indimethylformamide using triethylamine as the base. Under theseconditions, the 20-OH position is unaffected. For forming 10-O-esters,the acid chloride of the chosen reagent is used. In each case, thesequence of steps, after 10-hydroxy derivatization, described inExamples 1-5 is followed to generate 10-protected versions of CL1-SN-38, CL2-SN-38, CL-3-SN-38, CL4-SN-38, and CL5-SN-38, respectively.For the simplest case of R being ethoxy, the first step is theconversion of SN-38 to its 10-O-ethyl carbonate by treatment with ethylchloroformate in dimethylformamide using triethylamine as the base.

Example 7 Conjugation of maleimide-containing SN-38 intermediates tomildly reduced antibodies: attachment to interchain region of MAbs

The anti-CEACAM5 humanized MAb, hMN14, the anti-CD22 humanized MAb,hLL2, the anti-CD20 humanized MAb, hA20, the anti-EGP-1 humanized MAb,hRS7, and anti-MUC1 humanized MAb, hPAM4, were used in these studies.Each antibody was reduced with dithiothreitol (DTT), used in a50-to-70-fold molar excess, in 40 mM PBS, pH 7.4, containing 5.4 mMEDTA, at 37° C. (bath) for 45 min. The reduced product was purified oncentrifuged size-exclusion column and buffer-exchanged with 75 mM sodiumacetate-1 mM EDTA. The thiol content was determined by Ellman's assay,and was in the 6.5-to-8.5 SH/IgG range. Alternatively, the antibodiesare reduced with tris(2-carboxyethyl) phosphine (TCEP) in phosphatebuffer at pH in the range of 5-7, followed by in situ conjugation. Thereduced MAb was reacted with ˜10-to-15-fold molar excess of ‘CL1-SN-38’of Example 1, or ‘CL2-SN-38’ of Example 2, or ‘CL3-SN-38’ of Example 3,or ‘CL4-SN-38’ of Example 4, or ‘CL5-SN-38’ of Example 5, or‘CL2-SN-38-10-O—CO₂Et’ of Example 6, using DMSO at 10% v/v asco-solvent, and incubating for 20 min at ambient temperature. Theconjugate was purified by centrifuged SEC, passage through a hydrophobiccolumn, and finally by ultrafiltration-diafiltration. The product wasassayed for SN-38 by absorbance at 366 nm and correlating with standardvalues, while the protein concentration was deduced from absorbance at280 nm, corrected for spillover of SN-38 absorbance at this wavelength.This way, the SN-38/MAb substitution ratios were determined. Thepurified conjugates were stored as lyophilized formulations in glassvials, capped under vacuum and stored in a −20° C. freezer. SN-38 molarsubstitution ratios (MSR) obtained for some of these conjugates, whichare typically in the 5-to-7 range in view of the mode of conjugation,are shown in Table 2.

TABLE 2 SN-38/MAb Molar substitution ratios (MSR) in some conjugates MAbConjugate MSR hMN-14 hMN-14-[CL1-SN-38], using drug-linker of Example 17.7 hMN-14-[CL2-SN-38], using drug-linker of Example 2 6.8hMN-14-[CL3-SN-38], using drug-linker of Example 3 5.5hMN-14-[CL5-SN-38], using drug-linker of Example 5 6.9 hRS7hRS7-CL1-SN-38 using drug-linker of Example 1 5.3 hRS7-CL2-SN-38, usingdrug-linker of Example 2 6.3 hRS7-CL3-SN-38, using drug-linker ofExample 3 5.1 hPAM-4 hPAM-4-[CL2-SN-38], using drug-linker of Example 25.7 hLL2 hLL2-CL1-SN-38, using drug-linker of Example 1 7.4hLL2-CL2-SN-38, using drug-linker of Example 2 6.4 hA20 hA20-CL2-SN-38,using drug-linker of Example 2 6.1

Example 8 In vitro hydrolytic stabilities of different hMAb-SN-38conjugates: Fine-tuning of stability profiles by varying the linker andmodifying 10-hydroxy position

In vitro stabilities of SN-38 conjugates of anti-CEACAM5 antibody,hMN-14, derived from CL1-SN-38 of Example 1, CL2-SN-38 of Example 2,CL3-SN-38 of Example 3, CL4-SN-38 of Example 4, CL5-SN-38 of Example 5,and the 10-O-ethoxycarbonyl analog of CL2-SN-38, as described in Example6 were examined in 40 mM PBS at 37° C. At periodic intervals, aliquotswere withdrawn, a known amount of 10-hydroxycamptothecin used as aninternal standard was added, and the material was extracted by proteinprecipitation with acetonitrile, and extraction of SN-38 (dissociatedfrom antibody) by extraction with chloroform. Fixed volumes of theextracts were analyzed by reverse phase HPLC, quantifying for SN-38 byfluorescence detection of HPLC peaks. SN-38/internal standard peakratios were correlated with SN-38 standard curve, the latter generatedby plotting SN-38/internal standard peak ratios as a function of SN-38concentration. Plots of SN-38 dissociation kinetics were generated usingstandard Prism® software. FIG. 1 shows that steric hindrance around20-carbonate or 20-ester position in SN-38 enhances hydrolytic stabilityof the corresponding antibody conjugates. In addition, protection of10-hydroxy position of SN-38, as ethoxycarbonyl derivative, for instance(as shown in this Example), significantly enhances hydrolytic stability.Examples of 10-hydroxy protecting groups are enumerated in paragraph053. Thus, modulating the stability profiles of camptothecin conjugatesin general, and of SN-38 conjugates in particular, by variations inlinker design as described in these Examples, is also an embodiment ofthe present invention.

Example 9 In vitro cell-binding and cytotoxicity of antibody-SN-38conjugates in different cell lines

In vitro studies were conducted using LoVo human colon carcinoma cellsand anti-CEACAM5 antibody conjugates of SN-38 [hMN-14-(CL1-SN-38),hMN-14-(CL2-SN-38), and hMN-14-(CL3-SN38)], CaPan-1 human pancreaticcell line (for which anti-EGP-1 antibody hRS7 is positive) and Calu-3lung adenocacinoma cell line and hRS7 conjugates. In the latter two celllines, the conjugates used were hRS7-CL1-SN-38, hRS7-CL2-SN-38, andhRS7-CL3-SN-38. Cell lines were obtained from American Type CultureCollection (ATCC, Rockville, Md.). For cell-binding assays, unmodifiedantibodies were used as positive controls. In growth inhibition(cytotoxicity) assays, free SN-38 drug was used as positive control.Cell-binding to antigen-positive tissue culture cell lines was done byindirect cell surface binding ELISA assays. For growth inhibitionstudies, cells were harvested and plated into 96 well plates (25,000cells/well). 20 μL of serially diluted solutions of conjugates orcontrols were added to each well to final concentration of 0-7 μM finalconcentration of SN-38 equivalent, and incubated at 37° C. Totalincubation time was 96 h. MTS dye reduction assay was used to determinedose response curves, and effective EC₅₀ concentrations were determinedusing PrismPad® Software (Advanced Graphics Software, Encinitas,Calif.). Similarly, Capan-1 human pancreatic cell line was used forevaluation of SN-38 conjugates of anti MUC-1 antibody, hPAM4, which isspecific for this cell line. This shows preservation of cell-binding andcytotoxicity of SN-38 conjugates of hPAM-4. In a similar fashion,cell-binding and cytotoxicities of CL4-SN-38, CL5-SN-38, and10-ethoxycarbonyl analogs of CL1-SN-38, CL2-SN-38, CL3-SN-38, CL4-SN-38,and CL5-SN-38 conjugates of these antibodies show cell-bindings andgrowth inhibitions when these are examined in the cell lines for whichthe antibodies are specific.

The following figures demonstrate antigen-binding and growth inhibitingcapacity of exemplary conjugates. FIG. 2 shows binding of varioushMN14-SN38 immunoconjugates to the LoVo human colorectal adenocarcinomacell line. The CL-1, CL-2 and CL3 conjugates of SN38 to hMN14 showedbinding affinities (K_(d) values) that were comparable to theunconjugated hMN-14 IgG. FIG. 3 shows the in vitro cytotoxicity ofhMN14-SN38 immunoconjugates on the LoVo cell line. The CL1, CL2 and CL3SN38 conjugates of hMN14 exhibited comparable cytotoxicities(exemplified by IC₅₀ values) with free SN-38. The skilled artisan willrealize that in vivo the cytotoxicity of free SN-38 will be limited byits systemic toxicity, while the antibody-targeted delivery ofconjugated SN-38 will reduce systemic toxicity and allow highereffective doses of SN-38 to be delivered to the target cells or tissues.FIG. 4 illustrates cytotoxicity against the Calu-3 lung adenocarcinomacell line. The CL1, CL2 and CL3 conjugates of hRS7 showed EC₅₀ valuesthat were comparable with free SN38.

Example 10 In vivo therapy of lung metastases of GW-39 human colonictumors in nude mice using hMN-14-[CL1-SN-38] and hMN-14-[CL2-SN-38] withappropriate controls

A lung metastatic model of colonic carcinoma was established in nudemice by i.v. injection of GW-39 human colonic tumor suspension, andtherapy was initiated 14 days later. Specific anti-CEACAM5 antibodyconjugates, hMN14-CL1-SN-38 and hMN14-CL2-SN-38, as well as nontargetinganti-CD22 MAb control conjugates, hLL2-CL1-SN-38 and hLL2-CL2-SN38 andequidose mixtures of hMN14 and SN-38 were injected at a dose schedule ofq4dx8, using different doses. FIG. 5 (MSR=SN-38/antibody molarsubstitution ratio) shows selective therapeutic effects due to hMN-14conjugates. At equivalent dosages of 250 μg, the mice treated withhMN14-CL1-SN38 or hMN14-CL2-SN38 showed a median survival of greaterthan 107 days. Mice treated with the control conjugated antibodieshLL2-CL1-SN38 and hLL2-CL2-SN38, which do not specifically target lungcancer cells, showed median survival of 56 and 77 days, while micetreated with unconjugated hMN14 IgG and free SN38 showed a mediansurvival of 45 days, comparable to the untreated saline control of 43.5days. A significant and surprising increase in effectiveness of theconjugated, cancer cell targeted antibody-SN38 conjugate, which wassubstantially more effective than unconjugated antibody and freechemotherapeutic agent alone, was clearly seen. The dose-responsivenessof therapeutic effect of conjugated antibody was also observed. Theseresults demonstrate the clear superiority of the SN38-antibodyconjugates compared to the combined effect of both unconjugated antibodyand free SN38 in the same in vivo human lung cancer system.

Example 11 In vivo therapy of nude mice carrying CaPan1 human pancreatictumors using hPAM-4-[CL2-SN-38]

A s.c. pancreatic tumor model was established using human CaPan1 humantumor cells, and after the tumor volumes reached ˜0.2 cm³, therapy wasinitiated using the SN-38 conjugate of specific antibody, hPAM4, namely,hPAM4-CL2-SN-38. A dose schedule of q4dx8 using the protein dose of 0.5mg (drug dose of 0.39 mg of conjugated SN-38/kg of body weight) showedsignificant (treated vs. untreated week-3: P_(AUC)<0.01) tumor growthcontrol versus untreated (FIG. 6). All treated mice were alive >7 weeks,while all untreated mice were sacrificed by 41/2 weeks due to tumorburden. The mice treated with hPAM4-CL2-SN38 conjugated antibody showeda mean tumor volume of close to zero after 7 weeks (FIG. 6), while pointcontrol mice had a mean tumor volume of almost 2.0 cm³ after 4 weeks.The hPAM4-CL2-SN38 antibody conjugate was highly effective at reducingtumor burden and prolonging survival in this in vivo human pancreaticcancer model system.

Example 12 Elimination of HIV infection by treatment with a SN-38conjugate of an anti-gp120 MAb

A MAb targeted to the HIV envelope protein gp120, anti-gp120 antibodysuch as P4/D10, is reduced using conditions described in Example 7, andthe reduced MAb is reacted with a 20-fold molar excess of the druglinker CL2-SN-38, which is as described for Example 2. Ananti-gp120-SN-38 conjugate with a substitution of 8 drug molecules perantibody is obtained. An in vitro HIV-inhibition assay with saidconjugate is performed by using various mixtures of uninfected Jurkat-Tcells and fully HIV-infected Jurkat T-cells (in the ratios of 99.8:2 to95:5), and treating with serial dilutions of the conjugate, non-specifichRS7-CL-SN38 conjugate control, naked antibody, and HIV-negative serumfrom 100 to 0.00001 μg/mL. The cells so treated are incubated in RPMI1640 culture medium at 37° C. for seven days, and then assayed for HIVinhibition by the relevant ELISA test. This experiment shows a strongand specific inhibition of intercellular spread of HIV by the specificdrug conjugate. The in vivo efficacy is tested by administering micewith isologous HIV-infected cells together with specific andnon-specific SN-38 conjugates. For this, primary murine splenocytesinfected by HIV-1/MuLV pseudotype virus are intraperitoneallytransferred to groups of mice simultaneously with immunoconjugateadministration. Peritoneal cells are harvested 10 days later. Whileinfectious HIV presence is demonstrated in control mice, no infectiousHIV is detected in mice treated with 100 μg or less of anti-gp120-SN-38conjugate. No protection is seen with mice treated with controlconjugates.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

1. A method for treating a disease, comprising administering apharmaceutical composition to a subject, wherein the compositioncomprises a conjugate of a camptothecin drug and a targeting moiety ofthe formula: TM-[L3]-[L2]-[L1]_(m)AA_(n)-CPT, where TM is adisease-targeting moiety selected from the group consisting of a murine,chimeric, primatized, humanized, or human monoclonal antibody (MAb), andsaid antibody is in intact, fragment (Fab, Fab′, F(ab)₂, F(ab′)2), orsub-fragment (single-chain construct) form; CPT is camptothecin or ananalog thereof; L3 is a component of a cross-linker comprising anantibody-coupling moiety and one or more acetylene or azide groups; L2comprises a defined PEG with 1-12 monomeric units with azide oracetylene at one end, complementary to the acetylene or azide moiety inL3, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; L1 comprises a collapsible linker, or apeptidase-cleavable moiety optionally attached to a collapsible linker,or an acid-cleavable moiety; AA is an amino acid; m is an integer withvalues of 0 or 1, and n is an integer with values of 0, 1, 2, 3, or 4;and when n is >1, the amino acids represented by AA can be the same ordifferent.
 2. The method of claim 1, wherein L2 comprises a defined PEGwith 12 monomeric units.
 3. The method of claim 1, wherein L2 comprisesa defined PEG with 111 monomeric units.
 4. The method of claim 1,wherein L2 comprises a defined PEG with 10 monomeric units.
 5. Themethod of claim 1, wherein L2 comprises a defined PEG with 9 monomericunits.
 6. The method of claim 1, wherein L2 comprises a defined PEG with8 monomeric units.
 7. The method of claim 1, wherein L2 comprises adefined PEG with 7 monomeric units.
 8. The method of claim 1, wherein L2comprises a defined PEG with 6 monomeric units.
 9. The method of claim1, wherein L2 comprises a defined PEG with 5 monomeric units.
 10. Themethod of claim 1, wherein L2 comprises a defined PEG with 4 monomericunits.
 11. The method of claim 1, wherein L2 comprises a defined PEGwith 3 monomeric units.
 12. The method of claim 1, wherein L2 comprisesa defined PEG with 2 monomeric units.
 13. The method of claim 1, whereinL2 comprises a defined PEG with 1 monomeric unit.
 14. The method ofclaim 1, wherein the conjugate is administered in combination with oneor more other therapeutic modalities selected from the group consistingof unconjugated antibodies, radiolabeled antibodies, drug-conjugatedantibodies, toxin-conjugated antibodies, gene therapy, chemotherapy,therapeutic peptides, oligonucleotides, localized radiation therapy,surgery and interference RNA therapy.
 15. The method of claim 1, whereinthe disease is cancer, an infection with a pathogenic organism, or anautoimmune disease.
 16. The method of claim 1, wherein said antibody orantibody fragment is an IgG1, IgG2a, IgG3 or IgG4.
 17. The method ofclaim 1, wherein said MAb is an internalizing antibody.
 18. The methodof claim 1, wherein the antibody or fragment thereof binds to atumor-associated antigen (TAA).
 19. The method according to claim 18,wherein the TAA is selected from the group consisting of CD74, CD22,epithelial glycoprotein-1, carcinoembryonic antigen (CEA or CD66e),colon-specific antigen-p, alpha-fetoprotein, CC49, prostate-specificmembrane antigen, carbonic anhydrase IX, HER-2/neu, EGFR (ErbB1), ErbB2,ErbB3, ILGF, BrE3, CD19, CD20, CD21, CD23, CD33, CD45, CD74, CD80, VEGF,ED-B fibronectin, P1GF, a tumor angiogenesis antigen, MUC1, MUC2, MUC3,MUC4, gangliosides, HCG, EGP-2, CD37, HLA-DR, CD30, Ia, A3, A33, Ep-CAM,KS-1, Le(y), S100, PSA, tenascin, folate receptor, Thomas-Friedreichantigens, tumor necrosis antigens, Ga 733, IL-2, IL-6, T101, MAGE,migration inhibition factor (MIF), an antigen that is bound by L243, anantigen that is bound by PAM4, CD66a (BGP), CD66b (CGM6), CD66c (NCA),CD66d (CGM1) and TAC.
 20. The method of claim 15, wherein the autoimmunedisease is selected from the group consisting of immune-mediatedthrombocytopenias, acute idiopathic thrombocytopenic purpura and chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sjögren'ssyndrome, multiple sclerosis, Sydenham's chorea, myasthenia gravis,systemic lupus erythematosus, lupus nephritis, rheumatic fever,polyglandular syndromes, bullous pemphigoid, diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis ubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, rheumatoid arthritis, polymyositis/dermatomyositis,polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranousnephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis.
 21. The method of claim 15,wherein the infectious disease organism is selected from the groupconsisting of bacteria, rickettsia, mycoplasma, protozoa, fungi,viruses, parasites, human immunodeficiency virus (HIV), Mycobacterium oftuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes,West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellaabortus, rabies virus, influenza virus, cytomegalovirus, herpes simplexvirus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reo virus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae.
 22. The method of claim 15, wherein the cancer is selectedfrom the group consisting of testicular cancer, myeloid leukemia, B-celllymphomas, B-cell leukemias, chronic lymphocytic leukemia, T-celllymphomas, non-Hodgkin's lymphoma, Hodgkin's disease, prostate cancer,breast cancer, ovarian cancer, stomach cancer, bladder cancer,non-small-cell lung cancer, glioblastoma, colorectal cancer, pancreaticcancer, head and neck squamous cell carcinoma, multiple myeloma,melanoma, lung cancer, renal cancer, glioblastome multiforme,histiocytoma, myeloid leukemia, adenocarcinomas, sarcomas, glial tumorsand hepatocellular carcinoma.
 23. The method of claim 22, wherein saidMAb is selected from the group consisting of LL1, LL2, RFB4, hA20, 1F5,L243, RS7, PAM-4, MN-3, MN-14, MN-15, Mu-9, AFP-31, L19, G250, J591,CC49, L243, and Immu-31.
 24. The method according to claim 1, whereinsaid antibody-coupling moiety is thiol-reactive and is selected from thegroup consisting of maleimide, vinylsulfone, bromoacetamide, andiodoacetamide.
 25. The method according to claim 1 wherein said CPT isselected from the group consisting of CPT, 10-hydroxy camptothecin,SN-38, topotecan, lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin,and derivatives thereof.
 26. The method according to claim 25, whereinsaid CPT is SN-38.
 27. The method according to claim 16, wherein saidconjugate has a structural formula selected from the group consisting ofMAb-CL1-SN-38, MAb-CL2-SN-38, MAb-CL3-SN-38, MAb-CL4-SN-38,MAb-CL5-SN-38 and having a structure selected from:


28. The method according to claim 27, wherein the 10-hydroxy position ofSN-38 is protected as 10-O-ester or 10-O-carbonate derivative using a‘COR’ moiety where the R group is selected from substituted alkylresidue such as “N(CH₃)₂—(CH₂)_(n)—” where n is 2-10 and wherein theterminal amino group is optionally in the form of a quaternary salt forenhanced aqueous solubility, or simple alkyl residue such as“CH₃—(CH₂)_(n)—” where n is 0-10, or alkoxy residues such as“CH₃—(CH₂)_(n)—O—” where n is 0-10 or “N(CH₃)₂—(CH₂)_(n)—O—” where n is2-10, or “R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyland n is an integer with values of 0-10.
 29. The method according toclaim 1, wherein the cleavable polypeptide is selected from the groupconsisting of Phe-Lys, Val-Cit, Ala-Leu, Leu-Ala-Leu, andAla-Leu-Ala-Leu.
 30. The method according to claim 1, wherein said MAbis multispecific, with multiple binding arms to target at least twodifferent antigens or epitopes contained on the target cell or pathogen,and one or more targeting arms are conjugated to CPT.
 31. The methodaccording to claim 30, wherein said multispecific MAb is a bispecificand/or bivalent antibody construct comprising one or more antibodiesselected from the group consisting of LL1, LL2, hA20, IF5, L243, RS7,PAM-4, MN-14, MN-15, Mu-9, L19, G250, J591, CC49 and Immu-31.
 32. Themethod of claim 30, wherein said multispecific antibody binds to two ormore antigens selected from the group consisting of CD74, CD22,epithelial glycoprotein-1, carcinoembryonic antigen (CEA or CD66e),colon-specific antigen-p, alpha-fetoprotein, CC49, prostate-specificmembrane antigen, carbonic anhydrase IX, HER-2/neu, BrE3, CD19, CD20,CD21, CD23, CD33, CD38, CD40, CD44, CD45, CD79a, CD79b, CD80, CD133,CD138, VEGF, EGF receptor (ErbB1), ErbB2, ErbB3, P1GF, VEGF, ED-Bfibronectin, MUC1, MUC2, MUC3, MUC4, Tag-72, ILGF, gangliosides, HCG,EGP-2, CD37, HLA-DR, CD30, Ia, A3, A33, Ep-CAM, KS-1, Le(y), S100, PSA,tenascin, testis antigen, folate receptor, Thomas-Friedreich antigens,tumor necrosis antigens, tumor angiogenesis antigens, MIF, Ga 733, IL-2,IL-6, T101, MAGE, an antigen that is bound by L243, an antigen that isbound by PAM4, CD66a (BGP), CD66b (CGM6), CD66c (NCA), CD66d (CGM1),TAC, and cancer stem-cell antigens.
 33. The method of claim 1, whereinthe targeting moiety is a fusion protein.
 34. The method of claim 33,wherein the fusion protein is constructed by the ‘dock and lock’technology.